Protein synthesis by the ribosome directly couples genotype to phenotype in the cell, and its regulation is central to cellular physiology. Although many steps of translation have diverged since the last common ancestor, translation initiation and ribosome recycling remain intimately coupled in both bacteria and humans. These coupled events in the translation cycle are the focus of the present application. Our understanding of the molecular mechanism of protein synthesis has undergone a revolution in the last decade, built on rapid advances in the structural biology of the ribosome. High-resolution structures are now available for the entire ribosome in both bacteria and eukaryotes, and of the large ribosomal subunit in archaea. However, important questions relating to dynamic events in translation remain unanswered due to the challenge of isolating structural intermediates. In this application, we propose to probe the molecular mechanism of ribosome recycling in bacteria, a process catalyzed by the GTPase elongation factor G and a validated target of antibiotics. We will build on our prior groundbreaking results in this area by combining x-ray crystallography, cryo-electron microscopy (cryo-EM) and single-molecule biophysics. By using x-ray crystallography, cryo-EM, and new methods and tools we have developed to study human translation initiation factor eIF3, we will also probe human translation initiation, one of the most dynamic steps in protein synthesis. We will use our unique system to functionally reconstitute human translation initiation factor eIF3, cryo-EM, genetics in Neurospora crassa, and revolutionary new methods in genome engineering to dissect the contribution of eIF3 to start codon selection on human cellular mRNAs. The combination of eIF3 reconstitution in E. coli, genetics in N. crassa, mutagenesis in human cells, and cutting-edge cryo-EM provides a powerful and unique means to unravel the molecular contributions of eIF3 to translation initiation in humans. Taken together, the three aims of this application build on the fundamental insights into ribosome structure and function obtained in the prior funding period, and address key mechanisms in translational control that could be exploited for the development of new antibiotics and therapeutics.